1. Field of the Invention
The invention in the field of chemistry and biochemistry relates to the synthesis and use of novel polyamine transport (PAT) inhibitor compounds with pharmacological or agricultural uses and as probes for biochemical assays or for purification of selected polyamine binding targets. As drugs, these compounds are used to treat disorders of undesired cell proliferation, primarily cancer, alone or combined with other agents such as polyamine synthesis inhibitors. An assay employing some of these compounds is useful for monitoring polyamine uptake or transport (PAT) and allows analysis of binding sites for polyamines or for other basic ligands on a variety of molecules. This invention also relates to the synthesis and use of novel polyamine combinatorial libraries. These libraries are used to discover compositions that inhibit PAT and/or that bind to a cellular polyamine transporter (PATr). Various members of these libraries or compounds discovered through use of the libraries have utility as drugs, agricultural chemicals, and as probes. This invention also identifies key elements that comprise the polyamine binding sites of membrane as well as soluble proteins.
2. Description of the Background Art
Polyamines are ubiquitous molecules that provide a "buffer" system for the cell by modulating the activities of proteins, RNA, DNA, and lipids. Polyamines may play a direct role in apoptosis. Mammals and other organisms have an active polyamine uptake and recycling system that complements their polyamine synthetic capabilities. Because polyamines modulate such a large range of molecules and cellular activities, polyamine analogues, as disclosed herein, offer novel approaches for targeting a variety of disease states, particularly cancer, and also provide unique tools to monitor cellular activities.
Polyamines and Cancer
The potential of polyamines as anticancer agents has been long recognized. Polyamines affect chromatin structure in eukaryotes and prokaryotes by binding specifically to DNA (Balasundaram, D. et al., Mol. Cell. Biol. 100:129-140, 1991) so that condensation occurs when the binding sites on DNA are saturated. Acetylation of polyamines and histones lowers their affinity for DNA and is believed to occur in tandem to alter the structure and function of the nucleosome, thus regulating DNA replication and transcription by loosening DNA at the ends of the core particle. Because polyamines are absolutely essential for DNA replication, they are of interest in the treatment of cancer. Particular interest has been focused on preventing cell proliferation by lowering intracellular polyamine levels. Polyamine analogues as described herein are useful for preventing or treating cancer and other proliferative diseases by acting at a number of different levels. The present invention focuses on the inhibition of PAT. Other targets include induction of spermine/spermidine acetyltransferase (SSAT), hypusine modification, and other proteins that inhibit the cell cycle or induce apoptosis.
The Polyamine Transporter (PATr)
The increased demand for polyamines by rapidly growing, transformed cancer cells is only partially met by an increased rate of synthesis. To exploit this increased need for polyamines, synthesis inhibitors have been sought. Additionally, lowering polyamine concentrations can result in aberrations in chromatin structure leading to cell death or inhibition of proliferation (Quemener, V. et al., Anticancer Res. 14:443-448, 1994; Porter, C. W. et al., Cancer Res. 53:581-586, 1993). It has become increasingly apparent that the initial disappointing results observed in the clinic with polyamine synthesis inhibitors arises from compensatory increases in transport of polyamines by a specific active transport system (Seiler, N. et al., Int. J. Biochem. 22:211-218, 1990; Seiler, N. et al., J. Biochem. Cell. Biol. 28:843-861, 1996). The promising results observed in cell culture with a suicide substrate inhibitor of ornithine decarboxylase, .alpha.-difluoromethylornithine (DFMO), or with an inhibitor of S-adenosylmethionine decarboxylase, methylglyoxal bis(guanylhydrazone) (MGBG) did not transfer to human clinical trials (Schecter, P. J. et al., In Inhibition of Polyamine Metabolism. Biological Significance and Basis for New Therapies; McCann, P. P. et al., eds; 1987, pp 345-364). Since the only two avenues for carbon transfer into polyamine pools are synthesis or transport, simultaneous inhibition of both of these pathways is considered by the present inventors to be a promising anti-cancer therapeutic approach.
A study confirming the validity of this chemotherapeutic approach used transplanted murine L1210 leukemia cells that were deficient in PAT. Mice transplanted with the wild-type L1210 cancer cells (with intact PAT) died after 12 days, even when treated with DFMO. In contrast, DFMO mice transplanted with PAT-deficient L1210 cells lived longer than 60 days (Ask, A. et al., Cancer Lett. 66:29-34, 1992). These authors also showed that treatment of mice harboring wild-type L1210 cells with a combination of (1) DFMO (2) a low polyamine diet and (3) antibiotics (which decrease polyamine production by gut flora) resulted in prolonged survival compared to treatment with DFMO alone.
Augmented PAT into cancer cells promotes cell killing. J. L. Holley et al. (Cancer Res. 52:4190-4195, 1992) showed up to a 225-fold increase in cytotoxicity of a chlorambucil-spermidine conjugate compared to chlorambucil alone. A series of nitroimidazole-polyamine conjugates were also effective (Holley, J. L. et al., Biochem. Pharmacol. 43:763-769, 1992). Others showed that mice infected with a multi-drug resistant strain of malaria were cured by treatment with a chloroquinoline-putrescine conjugate (Singh, S. et al., J. Biol. Chem. 272:13506-13511, 1997). Thus, the effectiveness of cytotoxic compounds could be enhanced by their conjugation with polyamines. These effects may have been due to the exploitation of the PAT system to deliver these compounds into cancer cells. The present invention is therefore directed in part to rapid and efficient testing of many different conjugates between polyamines and known drugs for their transport into cells. Furthermore, as described below, this invention combines the cytotoxic properties of known drugs with the facilitated transport of polyamines, which relies on the present inventors' discoveries surrounding the PATr described herein. By accessing the database of structure-activity-relationships (SARs) of PATr substrates, the present inventors are able to predict the transportability of a novel chemical entity or a novel polyamine conjugate.
Polyamine Transport (PAT) Assays
There is no known high-throughput assay for measuring PAT. A radiochemical assay is used for biochemical analysis of transport and has been used to study PAT in yeast and a variety of mammalian cells (Kakinuma, Y. et al., Biochem. Biophys. Res. Comm. 216:985-992, 1995; Seiler, N. et al., Int. J. Biochem. Cell Biol. 28:843-861, 1996). See, for example Huber, M. et al. Cancer Res. 55:934-943, 1995.
The radiometric assay uses radiolabeled polyamines such as putrescine, spermidine or spermine, but, due to the low signal, large numbers of adherent or non-adherent cells are required. Additional care is required with spermine due to its non-specific adsorption to cells and plastics. Cells are mixed with the test compounds and the radiolabeled polyamine to initiate the assay. The cells are incubated for 1-60 minutes, depending on cell type. The assay is terminated by removal of the medium and cooling the plates to 4.degree. C. The cells are then washed with cold medium three times, dissolved in 0.1% sodium dodecyl sulfate and the radioactivity in solution is then determined by scintillation counting. This assay is difficult to scale up to a high throughput procedure due to the low signal from the radiolabel and the handling requirements inherent in procedures with radioactivity.
Combinatorial Approaches to Polyamines and Analogues
Combinatorial chemistry, a rapidly changing field of molecular exploration, is still in its infancy. For reviews, see Lam, K. S., Anticancer Drug Des. 12:145-167, 1997; Salemme, F. R. et al.; Structure 5:319-324, 1997; Gordon, E. M. et al., J. Med. Chem. 37:1385-1401, 1994; Gallop, M. A. et al., J. Med. Chem. 37:1233-1251, 1994). The pharmaceutical industry, is now realizing that the original approach of the combined synthesis of hundreds to thousands of compounds in one "flask" followed by testing and deconvoluting the results is a tedious process with many pitfalls. The more traditional approach of medicinal chemistry, that is, the synthesis and testing of one compound at a time, yields more reliable and informative results about the structure-activity relationship (SAR) around a target. The trend in combinatorial chemistry is therefore towards synthesis of multiple compounds at once, with each in a separate container. Therefore, many have adopted this one-compound/one-well parallel synthetic approach to molecular exploration. While many lead compounds have been generated this way, the chemistries do not necessarily lead to a molecule with the necessary drug-like characteristics.
Combinatorial chemistry, a rapidly changing field of molecular exploration, is still in its infancy. For reviews, see Lam, K. S., Anticancer Drug Des. 12:145-167, 1997; Salemme, F. R. et al.; Structure 5:319-324, 1997; Gordon, E. M. et al., J. Med. Chem. 37:1385-1401, 1994; Gallop, M. A. et al., J. Med. Chem. 37:1233-1251, 1994). The pharmaceutical industry, is now realizing that the original approach of the combined synthesis of hundreds to thousands of compounds in one "flask" followed by testing and deconvoluting the results is a tedious process with many pitfalls. The more traditional approach of medicinal chemistry, that is, the synthesis and testing of one compound at a time, yields more reliable and informative results about the SAR around a target. The trend in combinatorial chemistry is therefore toward synthesis of multiple compounds at once, with each in a separate container. Therefore, many have adopted this one-compound/one-well parallel synthetic approach. While many lead compounds have been generated this way, the chemistries do not necessarily lead to a molecule with the necessary drug-like characteristics.
Polyamine analogues are notoriously difficult to synthesize. Due to the polycationic nature of the final products, traditional chromatographic techniques such as silica gel chromatography cannot be used. Intermediates need a lipophilic protecting group that enables purification of the compounds and extraction with organic solvents. Bergeron has solved some of these problems through the use of the mesityl-type amino protecting group (Bergeron, R. J. et al., J. Med. Chem. 40:1475-1494, 1997), which not only solved the problems of handling (allowing purification by silica gel and extraction by organic solvents), but also gave a synthetic handle to extend the backbone of the polyamine. After treatment with NaH, a sodium amide anion is produced which can be alkylated with an alkyl halide to extend the backbone.
Although this approach extends synthetic possibilities somewhat, it is still significantly limited. Use of the mesityl group requires that a harsh reagent like HBr/HOAc be employed for removal, thereby limiting the substituents of the resulting polyamine acid-stable ones. The availability of suitable alkyl halides, together with the amino starting materials, is also limited. Therefore, while this approach has made significant inroads toward simpler analogue production, it is severely limited in its potential for structural diversity. Other synthetic approaches suffer from similar limitations (Moya, E. et al., In Neuropharmacology of Polyamines; Carter, C., ed.; Academic Press: London, 1997; pp. 167-184). Several initial reports of solid phase synthesis of polyamine analogues (Byk, G. et al., Tetrahed. Lett. 38:3219-3222, 1997; Furka, A., Int. J. Peptide Protein Res. 37:487, 1991) have serious limitations including a covalently attached linker residue and the lack of sufficient diversity of structural components. These deficiencies are effectively addressed by the present invention.
Induction of Spermine/Spermidine Acetyltransferase (SSAT)
Cellular levels of polyamines are tightly regulated so that only a small window of variability in concentration is tolerated. This regulation is mediated by the control of polyamine synthesis, uptake and catabolism. Abnormally high concentrations of polyamines induce the enzyme SSAT which is associated with apoptosis (Parchment, R. E. et al., Cancer Res. 49:6680-6686, 1989). Polyamine analogues induce apoptosis by induction of this enzyme (Ha, et al., Proc. Natl. Acad. Sci. 94:11557-11562, 1997; Albanese, L. et al., Biochem. J. 291:131-137, 1993) in a cell type-specific way, presumably due to the accumulation of the polyamine analogue in the cell and its binding to a polyamine sensitive repressor or activator of SSAT transcription. Acetylated polyamines, the products of the SSAT-catalyzed reaction, are substrates for the enzyme polyamine oxidase which generates stoichiometric release of H.sub.2 O.sub.2 believed to be responsible a more proximate cause of the apoptotic response.
Hypusine
The protein eIF-5A appears to play a role in protein synthesis, although its exact function remains obscure (Hanauske-Abelm, H. M. et al., FEBS Lett. 266:92-98, 1995). EIF-5A is unique in that it is modified by the unusual amino acid hypusine. Hypusine is generated post-translationally by the sequential action of deoxyhypusyl synthase (using spermidine as a substrate) and deoxyhypusyl hydroxylase. Inhibition of this modification of eIF-5A coincides with proliferative arrest late in the G1 phase of the cell cycle. This modification occurs in most, if not all, eukaryotes. The present inventors have noted that inhibitors of deoxyhypusyl synthase would be useful in treating diseases associated with unwanted cell proliferation, such as cancer, by blocking the cell cycle.
Inhibition of Angiogenesis
Inhibition of polyamine synthesis decreases the vascularization of solid tumors. One month of treatment with DFMO resulted in a 50% reduction in neoplastic vessel count in humans with cervical interepithelial neoplasia (Mitchell, M. F. et al., Proceedings AACR 39:Ab. 600, 1998). DFMO inhibited the neovascularization induced by tumor cells in vivo (Jasnis, M. A. et al., Cancer Lett 79:39-43, 1994). Squalene, a polyamine analogue, also inhibits angiogenesis in the rabbit cornea assay.
Other Mechanisms that Block Cell Growth or Induce Apoptosis
Transport, SSAT, and deoxyhypusine synthase are targets for developing therapies for cancer and other proliferative diseases. The potential polyamine related targets associated with cancer have not been exhausted. CHENSpm is polyamine analogue that induces apoptosis but does not function through any of the mechanisms described above (Ha, H. C. Proc Nat. Acad. Sci USA 94:11557-11562 (1997) CHENSpm does not induce SSAT, but does reduce spermidine and spermine levels and produces a G.sub.2 cell cycle arrest at subtoxic concentrations, suggesting an unusual mode of action. Polyamines are known to bind to tubulin and promote its bundling, though this is just one of several possible mechanisms by which polyamines can induce apoptosis or inhibit cell growth. For example, some microtubule associated proteins (MAPs) also bind polyamines.
Monitoring of Cancer-Related Molecules by Polyamine Analogues
Several polyamine binding anti-cancer targets could be monitored using various polyamine analogues. Acetylated polyamines, the products of the spermine/spermidine acetyltransferase (SSAT) enzymatic reaction, are substrates for the enzyme polyamine oxidase. Oxidation of acetylated polyamines produces a stoichiometric release of H.sub.2 O.sub.2 which is believed to be responsible for the apoptotic response. This induction is cell type-specific and is believed to be due to the accumulation of the polyamine analogue in the cell and its possible binding to a polyamine-sensitive repressor or activator of transcription of SSAT. This repressor has not been identified, but a probe/assay for its detection would enable the synthesis of better drugs.
Membrane-bound Proteins
Several cellular receptors have polyamine binding sites that influence receptor binding activity. Hypertension, osteoporosis, Alzheimer's disease and ischemia may all be targeted through polyamine binding receptors such as calcium receptor, N-methyl-D-aspartate (NMDA) receptors, glutamate receptors, Ca.sup.2+ channels and several of the inwardly rectifying K.sup.+ channels (Ventura, C. et al., Am. J. Physiol. 267 H587-H592, 1994).
Polyamines in Other Diseases
Post-Angioplasty Injury
Because PAT inhibitors can contribute to inhibition of cell growth, they are viewed by the present inventors as being useful in the treatment of post-angioplasty injury. Endothelial denudation and vessel wall injury lead to neointimal hyperplasia and luminal stenosis. Inhibition of smooth muscle cell proliferation, for example, could inhibit neointimal formation. According to this invention, this initiation of cell proliferation after injury is amenable to treatment with PAT inhibitors preferably in combination with polyamine synthesis inhibitors (Takagi, M. M. et al., Arterioscler. Thromb. Vasc. Biol. 17:3611-3619, 1997; Nakaoka, T. et al., J. Clin. Invest. 100:2824-2832, 1997; Maillard, L. et al., Cardiovasc. Res. 35:536-546, 1997).
Hypertension
Ca.sup.2+ channels, which have high affinity binding sites for polyamines, are modulated by polyamine levels. Polyamines modulate the .beta.-adrenergic-mediated changes in Ca.sup.2+ levels and contractility (Ventura, C. et al., Am. J. Physiol. 267: H587-H592, 1994). Ca.sup.2+ channels and binding can be measured as described for the NMDA receptor and the K.sup.+ -inward rectifying channels in Ventura, supra. Thus, an appropriate polyamine or analogue can be harnessed to modulate Ca.sup.2+ in place of the channel blockers currently in use.
Osteoporosis
Blood and tissue (e.g., nerve) calcium levels are modulated by the external Ca.sup.2+ sensing receptor (CaR) located in the parathyroid and kidney. The CaR has a specific polyamine binding site. Modulation of this receptor is believed to be a promising approach to the treatment of osteoporosis.
Alzheimer's Disease
The CaR plays a different role in the brain from that in the parathyroid. Aggregated .beta.-amyloid protein in this disease can stimulate the CaR and eventually lead to its down-regulation. Polyamines, likewise, can bind to the CaR and inhibit CaR down-regulation stimulated by .beta.-amyloid. Polyamines or polyamine analogues can therefore serve as protective molecules.
Immunosuppression
Low dose methotrexate is a common treatment for rheumatoid arthritis (RA). The reason for its efficacy is unknown, although it is not believed to inhibit proliferation of lymphoid cells. S-adenosylmethionine (AdoMet) metabolism has been proposed to play a direct role in its immunosuppressive activity.
The direct effect of AdoMet metabolism on the immune response is not known, though a role for polyamines has been suggested (Furumitsu, Y. et al., J. Rheumatology, 20:1661-1665, 1993; Nesher, G. et al., Arthr. Rheumat. 33:954-957, 1990). Cytokines are believed to play a direct role in the pathogenesis of RA, and IL-2 is low in patients' synovial fluid, a condition which was reversed by inhibitors of polyamines (Flesher, E. et al., J. Clin. Invest. 83:1356-1362, 1987). The polyamine synthesis inhibitor DFMO prolongs the life of MRL-lpr/lpr mice, a model of systemic lupus erythematosus.
Polyamine levels are elevated in the urine, synovial fluid, synovial tissue, and peripheral blood mononuclear cells of RA patients. Culturing these cells in the presence of methotrexate inhibited the production of IgM-rheumatoid factor. Spermidine reversed this effect, indicating to the present inventors that a combination of polyamine synthesis inhibitors and PAT inhibitors are useful can treat autoimmune diseases. Other polyamine analogues, spergualin and deoxyspergualin, are immunosuppressive and may be beneficial for treating multiple sclerosis (Bergeron et al., J. Org. Chem. 52:1700, 1987; Drug Fut. 16:1165, 1991).
Psychiatric Disorders
A number of the compounds which the present inventors have found to inhibit PAT with high affinity, as disclosed herein, structurally resemble several known antipsychotic or antidepressant drugs.
DNA/RNA-Polyamine Hybrids for Stable Binding to Nucleic Acids
The spacing between ammonium polycations in naturally occurring polyamines (spermidine and spermine) is 3-4 carbons. This is the exact spatial separation for optimal binding to a DNA or RNA polyanionic phosphate backbone. It has been suggested that this interaction, together with the interaction with chromatin proteins, modulates gene transcription and expression. Recently, such ionic interactions have been exploited by combining polycationic 3', 5'-polyguanidine linkers bound with the base portion of the nucleosides to enhance double or triple helix formation. An oligomeric polyadenosyl RNA analogue in which the phosphodiester backbone units were replaced by cationic guanidine units was inactive, since triple strand formation was decreased (Dempey, R. D. et al., Proc. Natl. Acad. Sci. U.S.A. 93:4326-4330, 1996; Goodnow, Jr., R. A. et al., Tetrahedron Lett. 38:3195-3198 and 3199-3202, 1997). These nucleic acid analogues melt like double helices consistent with Watson-Crick base pairing.
In this regard, this invention provides compositions and methods that incorporate complex structural substituents onto a polyamine chain to optimize the targeting of DNA or RNA for inhibiting replication, transcription or translation.
Other Polyamine-Binding Receptors as Targets
Of particular pharmacological interest are the ways in which polyamines modulate various receptor or channel functions. In particular, polyamines modulate the Ca.sup.+ -permeable glutamate receptors assembled from subunits containing a glycine residue at the RNA editing site. The inward rectification of the K.sup.+ inward rectifying channels is induced by blocking the outward current using cytoplasmic Mg.sup.2+ (or intrinsic channel gating). This gating is due primarily to a block by cytoplasmic polyamines (Shyng-Si, et al. Proc. Natl. Acad. Sci. USA. 93:12014-12019, 1996). According to this invention, polyamine analogues are useful for modulating glutamate receptors that are important in ischemia, strokes, and cardiovascular disease. NMDA receptor antagonists act as anticonvulsants so that agents active at NMDA receptors are additional useful targets.
Anti-Infective Effects of Polyamine Modulation
Parasitic organisms such as Trypanososma cruzi are thought to obtain the polyamines necessary for their growth from their hosts rather than synthesize their own. DFMO (an ODC inhibitor), decreases the availability of putrescine, a precursor of spermidine and spermine synthesis. DFMO can cure T. brucei infection in mice and is active against African sleeping sickness in humans caused by T. brucei gambiense. DFMO also has clinical utility in Pneumocystis carinii pneumonia and in infection by the coccidian protozoan parasite, Cryptosporidium. In the laboratory, DFMO acts against Acanthamoeba, Leishmania, Giardia, Plasmodia and Eimeria (Marton, L. J. et al., Annu. Rev. Pharmacol. Toxicol. 35:55-91, 1995). Polyamines are also essential for the growth of Hemophilus and Neisseria organisms (Cohen, S. S., A Guide to the Polyamines, Oxford University Press, NY. pp 94-121, 1998). Thus, compounds and methods of the present invention can be used to treat diseases caused by Trypanososma cruzi, T. brucei, Pneumocystis carinii, Cryptosporidium, Acanthamoeba, Leishmania, Giardia, Plasmodia, Eimeria, Hemophilus and Neisseria.
Plant Pathogens
Lowering of polyamine levels may protect plants against a wide range of fungi, e.g., Uremyces phaseoli Linnaeus, race O. Unifoliolate (bean rust). DFMO is an effective fungicide in the following plants: tomato plants against Verticillium wilt fungus; wheat against stem rust fungus and powdery mild fungus; bean plants against powdery mildew fungus; Macintosh apple leaves against the powdery mildew fungus; Ogle oats against leaf rust fungus; and corn against the corn rust fungus (U.S. Pat. No. 4,818,770). The compositions of this invention that lower polyamine levels by PAT inhibition or that have other actions on systems that utilize, or are affected by, polyamines could be useful in protecting plants against a wide range of fungi.
Miscellaneous Targets
Polyamines may protect DNA from radiation damage (Newton, G. L. et al., Radiat. Res. 145:776-780, 1996). Therefore, an agent that raises polyamine levels, or that substitutes for an endogenous polyamine more effectively, may be a useful adjunct to radiotherapy. Polyamines may play an important role in controlling mammalian fertility (U.S. Pat. No. 4,309,442) and maintaining embryonic growth (U.S. Pat. No. 4,309,442). Other actions ascribed to polyamines include anti-diarrheal, anti-peristaltic, gastrointestinal anti-spasmodic, anti-viral, anti-retroviral, anti-psoriatic and insecticidal (U.S. Pat. No. 5,656,671).
The present invention also has use in: (a) cleanup of toxic or radioactive metal waste that requires specific ion-binding (this can be designed into a polyamine for use in vivo or environmentally); (b) image-enhancement in medical imaging systems such as X-ray, computer-assisted tomography or magnetic resonance technologies; (c) enzyme-like catalysis that is required in asymmetric organic synthesis or resolution; (d) xenobiotic detoxification; and (e) nucleosidase activities.
Combination Therapy
Certain agents inhibit growth of tumor cells in culture in a manner that is additive with cytotoxic drugs. The agent 8-chloro-cAMP (8-Cl-cAMP) (Tortora et al., Cancer Res. 57:5107-5111, 1997) is a cAMP analogue that selectively down-regulates PLA-1, a signaling protein that is directly involved in cell proliferation and neoplastic transformation and that mediates the mitogenic effects of certain oncogenes and growth factors. In nude mice bearing human GEO colon cancer xenografts, 8-Cl-cAMP inhibited tumor angiogenesis and secretion of growth factors of the EGF family and synergized with anti-EGF receptor antibodies in inhibiting tumor growth. 8-Cl-cAMP acts by different mechanisms than do the polyamine analogues of this invention.
The PAT inhibitors of the present invention can be used alone, in combination with 8-Cl-cAMP, or in combination with an ODC inhibitor and/or a SAM decarboxylation inhibitor (with or without 8-Cl-cAMP). Because polyamine modulation affects chromatin structure, other agents can be used in combination with the PAT inhibitors of this invention include topoisomerase inhibitors, DNA alkylating agents and DNA intercalating agents such as doxorubicin, adriamycin, chlorozotocin, etc.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.